The overall objective for this project is to develop and implement a comprehensive set of technologies to improve screening and diagnosis of conditions associated with Fragile X Syndrome (FXS). FXS is caused by an expansion of a cytosine-guanine-guanine (CGG) triplet repeat in the 5'-untranslated region of the FMR1 gene. FXS affects 1/4000 men and 1/6000 women. Full expansion to greater than 200 repeats is associated with hypermethylation of the FMR1 gene and complete loss of FMR1 protein production. A more modest expansion of the CGG repeats is associated with Fragile X-associated Tremor/Ataxia Syndrome (FX-TAS) in older men and primary ovarian insufficiency (FX-POI) in women. Recently published guidelines suggest follow- up chromosome and Fragile X testing for a diagnosis of autism spectrum disorder (ASD), which impacts 1/150 individuals-roughly 33 times the population incidence of Fragile X. Furthermore, promising drugs are currently in clinical trials and will require accurate and early identification of Fragile X patients. Improvements in testing for Fragile X will have important implications for a broad range of individuals of all ages across multiple mental and health conditions associated with this disorder. Our phase I grant was focused on developing amplification technologies that can enable rapid, high throughput and sensitive detection of CGG repeat numbers, and methylation associated with FXS. Furthermore, these technologies would be able to address confounding zygosity in females, a feature that is not available on commercial PCR based FXS tests. All of the major milestones of phase I have been met and exceeded. We have developed three PCR technologies, some of them with overlapping functionality that can address all major issues associated with FXS testing. These technologies show promise for completely replacing the need for reflex testing with Southern blots, which are cumbersome and slow. In the phase II portion of this grant we propose studies to integrate the PCR technologies into comprehensive and streamlined test. This includes the development of a complete set of controls, quality control procedures and a computer interface for data analysis and storage. The comprehensive test workflows will be evaluated in archived clinical specimens. This phase II grant will result in a comprehensive diagnostic workflow that can support routine testing and screening, and thus enable earlier interventions and improved treatment options for patients. PUBLIC HEALTH RELEVANCE: The long-term goal for this project is to improve screening and diagnosis of Fragile X Syndrome and related conditions. In this project, we will leverage our breakthrough in PCR of the FMR1 gene to develop a set of robust and accurate tests that are cost-effective and efficient. This will enable widespread screening to identify carriers and permit earlier diagnosis and intervention for Fragile X Syndrome patients.